1. Field of the Invention
The invention relates to an apparatus and method for pathogen detection. Specifically, the invention relates to the field of pathogen detection systems and diagnostic devices and their micro-component assembly. More specifically, the invention utilizes an apparatus that includes a dielectrophoretic separator, a dielectrophoretic condenser, a dielectrophoretic trap, microfluidic components, and field effect sensor, such as an ion sensitive sensor, nanowire sensor, or nanoribbon sensor configured as biosensors, to perform a pathogen detection process.
2. Description of Related Art
Bacterial infections cause thousands of diseases in humans and animals every year. Recent deadly outbreaks of E. Coli, Salmonella, and Listeria have highlighted the urgent need for more effective methods of detection, identification, and characterization of pathogens, and their origin and proliferation. Conventional detection methods have proven inadequate because they suffer from long incubation periods, high cost, and require highly trained personnel to operate. There remains a strong need for a reliable, time-efficient apparatus and method for specific detection of bacteria in low concentration.
Conventional methods rely on bacterial culture growth, which require highly qualified personnel and time, both contributing to higher costs for the procedure. The most widely used method for bacterial detection, the standard plate count, takes from 24 to 48 hours due to the time needed for bacteria to grow detectable colonies, and requires a stocked microbiology lab. Although faster methods, such as PCR (Polymerase Chain Reaction) Plates or labeled detection and fluorescent imaging, can reduce the response time to one hour, these require complex sample preparation, highly trained personnel, high cost per test, and have limited portability.
The major challenge in automated sample preparation for detection from blood or other unprocessed liquids using microstructures is efficient separation of the analyte of interest (bacteria, cells, or particles) from large blood components. Red blood cells (RBC) and white blood cells (WBC) range between 6 μm-21 μm in size and constitute over 50% of the whole blood volume. RBC and WBC presence obstructs the detection of bacteria, cells, or particles. The present invention is a miniaturized device for rapid pathogen screening that overcomes these obstacles.
Dielectrophoresis (“DEP”) is a separation method based on size and dielectric properties and has been described in literature as for example in Pohl et al, Science 1966, and Sher Nature 1968, Voldman, Annual Review Of Biomedical Engineering, 2006. The use of DEP to manipulate particles and cells has been previously described, as for example, in H. Pohl, I. Hawk, “Separation Of Living And Dead Cells By Dielectrophoresis,” Science, 152, 3722 (1966); Y. Huang, R. Holzel, R. Pethig, X. Wang, “Differences In The Ac Electrodynamics Of Viable And Non-Viable Yeast Cells Determined Through Combined Dielectrophoresis And Electrorotation Studies,” Phys. Med. Bid., 37, 7 (1992); S. Chang, Y. Cho, “A Continuous Size-Dependent Particle Separator Using A Negative Dielectrophoretic Virtual Pillar Array,” Lab Chip, 8, 1930-1936 (2008); and J. Yang, Y. Huang, X. Wang, F. Becker, P. Gascoyne, “Differential Analysis Of Human Leukocytes By Dielectrophoretic Field-Flow-Fractionation,” Biophysical Journal, 78, 2680-2689 (2000). However, effective methods for cell/pathogen separation on a micro-scale from fluids containing pollutants of comparable size are still unattainable.
High-frequency electric fields when applied to an electrically neutral object cause polarization. A high-frequency non-uniform electric field gives rise to a dielectrophoretic force (DEP) FDEP which acts on the object.
A spherical object of a given electrical permittivity ∈p placed in a medium of a different permittivity ∈m in a spatially varying electric field E(x,ω) is subjected to a dielectrophoretic force, FDEP. The dielectrophoretic force is given by:FDEP=2π∈mr3Re{CM(ω)}·∇E2 whereCM(ω)=(∈{tilde over (p)}−∈{tilde over (m)})/(∈{tilde over (p)}+2∈{tilde over (m)}){tilde over (∈)}=∈+σ/iω;                 CM(ω) is the Clausius-Mossotti factor;        Re{CM(ω)} is the real part of the CM(ω), which can be a complex number;        ∈p is the particle permittivity;        ∈m is the permittivity of the liquid medium;        r is the particle radius;        {tilde over (∈)} is the complex permittivity (complex dielectric function);        σ is the conductivity;        i is the imaginary unit;        ω is the angular frequency; and        ∇E is the gradient of the electric field        
Depending on the respective permeability ({tilde over (∈)}) and conductivity (σ) of the object and the medium, the force can be attractive (positive dielectrophoresis (pDEP)), or repulsive (negative dielectrophoresis (nDEP)). If Re{CM(ω)} is positive, then the particle experiences a positive dielectrophoretic force, and if Re{CM(ω)} is negative, then the particle experiences a negative dielectrophoretic force. Different species have different dielectric properties. The dielectric functions ∈m, ∈p depend on the frequency of the external electric field. The permittivity of the medium affects the CM(ω) factor and the value of Re{CM(ω)}. Importantly, if the signs of Re{CM(ω)} for different species are opposite then the species are subject to forces acting in opposite directions and separation occurs.
There is a cross-over frequency, ωco, that occurs when the Re{CM(ω)} goes to zero. Critical to separation is that ωco is uniquely different for different cells and bacteria. Separation procedures for stained cells have been described in U.S. Pat. No. 7,153,648 entitled “Dielectrophoretic Separation Of Stained Cells,” where appropriate frequency and amplitude are applied via a function generator, and red blood cells are attracted to electrodes by positive dielectrophoresis force, while stained white blood cells are repelled to the area with weakest electric field by negative dielectrophoresis force. The differential behavior and separation of E. Coli cells from human blood cells on electrodes under applied electric field has been described in U.S. Pat. No. 6,989,086 entitled “Channel-less separation of bioparticles on an Electronic Chip by Dielectrophoresis.”
The dielectrophoretic force is affected both by the geometry of the electrodes (gradient of the electric field), the Re{CM(ω)} factor, and depends on the dielectric constant of the medium ∈m.
Using the aforementioned prior art techniques for dielectrophoresis, the separation of bacteria from blood may achieve, at best, an efficiency of approximately 30%.